Jia et al. BMC Infectious Diseases 2014, 14:506http://www.biomedcentral.com/1471-2334/14/506

RESEARCH ARTICLE Open Access

MicroRNA expression profile in exosomediscriminates extremely severe infections frommild infections for hand, foot and mouth diseaseHong-Ling Jia2†, Chun-Hui He1†, Zhuo-Ya Wang4, Yu-Fen Xu1, Gen-Quan Yin1, Li-Jia Mao1, Chao-Wu Liu3*

and Li Deng1*

Abstract

Background: Changes of miRNAs in exosome have been reported in different disease diagnosis and provided aspotential biomarkers. In this study, we compared microRNA profile in exosomes in 5 MHFMD and 5 ESHFMD as wellas in 5 healthy children.

Methods: Different expression of miRNAs in exosomes across all the three groups were screened using miRNAmicroarray method. Further validated test was conducted through quantitative real-time PCR assays with 54exosome samples (18 ESHFMD, 18 MHFMD, and 18 healthy control). The judgment accuracy was then estimated bythe receiver operating characteristic (ROC) curve analysis; and the specificity and sensitivity were evaluated by themultiple logistic regression analysis.

Results: There were 11 different miRNAs in exosomes of MHFMD and ESHFMD compared to healthy children, of which4 were up-regulated and 7 were down-regulated. Further validation indicated that the 4 significant differentially expressedcandidate miRNAs (miR-671-5p, miR-16-5p, miR-150-3p, and miR-4281) in exosome showed the same changes as in themicroarray analysis, and the expression level of three miRNAs (miR-671-5p, miR-16-5p, and miR-150-3p) were significantlydifferent between MHFMD or ESHFMD and the healthy controls. The accuracy of the test results were high with the undercurve (AUC) value range from 0.79 to 1.00. They also provided a specificity of 72%-100% and a sensitivity of 78%-100%,which possessed ability to discriminate ESHFMD from MHFMD with the AUC value of 0.76-0.82.

Conclusions: This study indicated that the exosomal miRNA from patients with different condition of HFMD expressunique miRNA profiles. Exosomal miRNA expression profiles may provide supplemental biomarkers for diagnosing andsubtyping HFMD infections.

Keywords: Exosomal microRNA Profile, HFMD, Diagnosis, Biomarker

BackgroundHand, foot, and mouth disease (HFMD) is a commonacute viral illness which has been epidemic worldwide[1-3]. The two major causative agents are known as hu-man enterovirus 71 (EV71) [4] and coxsackievirus A16(CVA16), which accounting for more than 70% of cases

* Correspondence: chwliu@hotmail.com; dr_dengli@163.com†Equal contributors3Guangdong Institute of Microbiology/State Key Laboratory of AppliedMicrobiology Southern China/Guangdong Provincial Key Laboratory ofMicrobial Culture Collection and Application, Guangzhou 510070,Guangdong, China1Guangzhou Women and Children’s Medical Center, Guangzhou 510120,Guangdong, ChinaFull list of author information is available at the end of the article

© 2014 Jia et al.; licensee BioMed Central Ltd.Commons Attribution License (http://creativecreproduction in any medium, provided the orDedication waiver (http://creativecommons.orunless otherwise stated.

in recent outbreaks [5]. Moreover, the effective and reli-able tool for diagnosing of HFMD is not available [6,7].The extremely severe HFMD (ESHFMD) mainly caused

by EV71 with severe neurologic clinical symptoms and sig-nificant fatalities [8-10], that had caused serious publichealth concerns. Many children with extremely severeHFMD were died before making a definite diagnosis. Thus,a rapid and reliable diagnostic method is essential for ap-propriate treatment and prophylaxis.Exosome represent a specific subtype of secreted mem-

brane vesicles that are approximately 30–100 nm insize and are formed inside the secreting cells in endosomalcompartments called multivesicular bodies [11,12]. Secreted

This is an Open Access article distributed under the terms of the Creativeommons.org/licenses/by/4.0), which permits unrestricted use, distribution, andiginal work is properly credited. The Creative Commons Public Domaing/publicdomain/zero/1.0/) applies to the data made available in this article,

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vesicles play an important role in normal physiologicalprocesses, development, and conditions such as viral in-fection [13-15] and are a possible source or pool ofnovel biomarkers of many diseases [16-18]. Exosomecontain proteins, miRNAs, and mRNAs, and the exoso-mal lipid bilayer protects this genetic information fromdegradation. Moreover, miRNAs can be transferred byan exosomal route and further exert gene silencing inrecipient cells [19,20], where they play an essentialregulatory role during development, with their levelschanging in different cell types and at different devel-opmental stages [21,22]. Although the mechanisticbasis for alterations in miRNA, especially in the contextof cellular malfunction, is not well understood, such al-terations play a pivotal role in pathological processesand have recently been proposed as biomarkers forbrain neoplasms, degenerative diseases, autism, andschizophrenia [23-25]. Changes in exosomal miRNAshave been reported in patients diagnosed with Alzheimer’sdisease (AD), and miRNAs have been shown to providediagnostic biomarkers [26].Here, we employed microarray methods to compare

the miRNAs of exosome from serum samples collectedfrom normal children and patients with mild HFMD(MHFMD) and extremely severe HFMD (ESHFMD). Wefocused on the miRNA profile of exosome and con-firmed whether these changes could be used to discrim-ination of specific condition for ESHFMD and MHFMD.

MethodsSerum sample preparationEthical approval was obtained for human sample collec-tion from the Ethics Committees at Guangzhou Womenand Children’s Medical Center, and written informedconsent was obtained from all guardians. Blood samplesfrom five MHFMD and five ESHFMD children diagnosedaccording to the Hand Foot and Mouth Disease PreventionControl Guide (2008 edition) issued by the Ministry ofHealth of China (http://www.moh.gov.cn/publicfiles/busi-ness/htmlfiles/mohbgt/s9511/200805/34775.htm) were ran-domly collected for 2-DE, and clinical symptoms andlaboratory testing (EV71 nucleic acid detection kit) con-firmed that EV71 infection caused HFMD in all these cases.In addition to meeting the above criteria, ESHFMD pa-tients all had encephalitis and pulmonary haemorrhage,required mechanical ventilation, and had other clinicalsymptoms. They were confirmed to have no other dis-ease after a systematic check in the hospital. Five bloodsamples from healthy children were collected as con-trols. To validate the miRNA microarray results, werandomly collected blood samples of 18 ESHFMD pa-tients and 18 MHFMD patients according to the diag-nostic guidelines described above and subjected thesamples to real-time quantitative RT-PCR. Another 18

blood samples from healthy children were collected ascontrols. Blood samples were separated by centrifuga-tion at 1,000 × g for 10 min. Serum aliquots were col-lected and stored at −80°C. The serum obtained wasfurther processed for exosome isolation.

ExoQuick precipitation of serum exosomeWe isolated exosome from the sera of all participants byusing ExoQuick precipitation (System Biosciences Inc,Mountain View, CA) following the manufacturer’s in-structions [27,28].

Exosome characterizationTransmission electron microscopy (TEM)The exosome extraction reagent was used to precipitatethe exosome from serum, which were then centrifugedat 1,500 × g for 10 min at 4°C to remove the supernatant.The exosome pellet was resuspended in 10 mM PBS infour times the volume of serum. A copper mesh wasplaced on a clean wax plate, and 100 μl of the exosomesuspension was added. After 4 min, the copper meshwas removed and placed in 2% phosphotungstic acid for5 min. The mesh was laid on the filter paper for air-drying, and TEM was used to observe the morphologicalfeatures of the exosome.

Western blot analysisThe exosome pellet was dissolved in the protein lysisbuffer, and the protein concentration was determinedusing a Bradford protein assay kit (Bio-Rad, USA). Sam-ples were separated on a 1D SDS-PAGE gel before trans-fer to a PVDF membrane. The membrane was incubatedwith the TSG101, CD63, CD9, HSP90α and Flotillin pri-mary antibodies at 4°C overnight, followed by incubationwith the corresponding secondary antibodies at roomtemperature for 1 h. Specific protein bands were visual-ized using the SuperSignal chemiluminescence system(ECL, Pierce, USA) and imaged by autoradiography.

RNA extraction from exosomeRNA was extracted from the exosome pellets using TRI-ZOL reagent according to the manufacturer’s protocol.Briefly, 1.0 ml of TRIZOL reagent and 200 μl of chloro-form were added to the sample, and the mixture wasvortexed for 60 s and allowed to stand at 25°C for5 min. After the mixture was centrifuged at 10,000 × gfor 10 min at 4°C, the supernatant was transferred to afresh tube and 500 μl of isopropanol was added. Afterincubation at −20°C overnight, the mixture was centri-fuged at 10,000 × g for 10 min at 4°C to remove thesupernatant, and the RNA pellet was washed with 75%ethanol. After ethanol removal by centrifugation at10,000 × g for 10 min at 4°C, the RNA was air-dried for5 min and then dissolved in 20 μl of RNase-free water.

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The purity of the isolated RNA was determined accord-ing to the OD260/280 using a Nanodrop ND-1000 sys-tem (Thermo Fisher Scientific).

Microarray analysisPooled exosome of serum from five healthy children, fiveMHFMD and five ESHFMD patients were used for miRNAmicroarray analysis. Total miRNA from these three pooledsamples was extracted as described above. Microarrayhybridization, data generation, and normalization were per-formed by the Shanghai Biochip Corp. following standardAgilent protocols. Human miRNA microarrays fromAgilent Technologies, which contain probes for 1,887human miRNAs from the Sanger database v.18.0, wereused in this study. Visualization of microarray data wasperformed using MeV 4.6 software (MultiExperimentViewer; http://www.tm4.org/mev.html). The microarraydata described herein have been deposited in the NationalCenter for Biotechnology Information Gene ExpressionOmnibus (http://www.ncbi.nlm.nih.gov/geo/), with acces-sion number GSE52780. A miRNA was designated asoverexpressed if expression in one of the pooled sampleswas >1.5-fold higher than that in another sample. In caseswhere overexpression was determined for a differentiallyexpressed miRNA, miRNA with the maximum intensityover 4 (log2 transformed intensity) in at least one of thepaired samples was considered.

Validation of real-time quantitative PCRFor testing of candidate miRNAs identified by microar-rays, real-time quantitative PCR (qRT-PCR) was per-formed using the Power SYBR Green PCR Master Mix(Applied Biosystems) in an ABI 7500 Real-Time PCRSystem (Applied Biosystems). The assays were per-formed on 54 samples (18 Control, 18MHFMD and 18ESHFMD) for four candidates (miR-16-5p, miR-15-3p,mir-4281, and miR-671) that met the defined criteria.Each reaction was performed in a 20 μl volume systemcontaining 5 μl of cDNA, 0.5 μl of each primer, 10 μl ofPower SYBR Green PCR Master Mix, and 4.0 μl ofRNase-free water. The PCR program consisted of de-naturation at 95°C for 2 min, followed by 40 cycles eachof denaturation for 15 s at 95°C and annealing and exten-sion for 30 s at 60°C. The miR-642a-3p expression levelwas used as a stable endogenous control for normalization.All assays were conducted in triplicate. miRNAs thatshowed cycle threshold values above 35 in one of the 54samples were excluded from additional statistical analysis.

Target prediction and enrichment informationThe target genes of the candidate miRNAs were predictedby TargetScan prediction software (http://www.targetscan.org/). The Gene Ontology (GO) and Kyoto Encyclopediaof Genes and Genomes (KEGG) database analyses were

conducted using a DAVID online analysis tool (http://david.abcc.ncifcrf.gov/), in which we focused on theGene Ontology (GO) biological processes feature. Foreach analysis, we used P < 0.05 as a cut-off.

Statistical analysisFor the qRT-PCR data, relative miRNA expression levelswere calculated by the comparative 2-△△Ct method as de-scribed previously [29]. Statistical significance was deter-mined using Student’s t-test. P < 0.05 was consideredstatistically significant. Receiver operating characteristic(ROC) curves were constructed to determine the specifi-city and sensitivity of individual miRNAs as surrogatebiomarkers. Area under the ROC curve (AUC) was usedas an accuracy index for evaluating the diagnostic per-formance of the selected miRNA panel. MedCalc (ver-sion 10.4.7.0; MedCalc, Mariakerke, Belgium) softwarewas used to perform the ROC analysis.

ResultsExosome isolation and validationMicrovesicles isolated from sera of controls and MHFMDand ESHFMD patients were assessed by TEM and westernblotting. TEM showed spherical structures approximately30–100 nm in diameter, consistent with previously re-ported characteristics of exosomes (Figure 1A). We fur-ther confirmed that these microvesicles were exosome byperforming western blot analysis on lysates using anti-bodies against five commonly used exosomal markers,TSG101, CD63, CD9, HSP90α and Flotillin. Levels ofTSG101, CD63, CD9, HSP90α and Flotillin were strik-ingly higher in the microvesicle fraction than in serum(Figure 1B). These results confirmed the identification andcharacterization of isolated microvesicles as exosomes.

Global exosome miRNA profiling from microarray analysisTo screen for candidate exosomal miRNAs fromMHFMD and ESHFMD patient serum samples, miRNAmicroarrays were used to evaluate the three groups(MHFMD, ESHFMD, and control). The microarray re-sults identified various miRNAs that were differentiallyregulated in the exosome of MHFMD and ESHFMDsamples relative to healthy controls, and a scatter plotwas generated (Figure 2A). Subsequently, we conductedpairwise comparison of the results of the scatter plotcharts and found 36 miRNAs with significantly differentexpression between MHFMD and the control, 68 miR-NAs differentially expressed between ESHFMD and thecontrol, and 65 miRNAs differentially expressed be-tween ESHFMD and MHFMD. Eleven miRNAs, thatare miR-671-5p, miR-4463, miR-144-3p, miR-4271,miR-4433-3p, miR-19b-3p, miR-4428, miR-135a-3p,miR-4281, miR-16-5p, and miR-150-3p, showed signifi-cantly different expression across all three groups

Figure 1 Characterization of exosomes in serum samples from healthy children and MHFMD and ESHFMD patients by transmissionelectron microscopy and western blotting. (A) Morphological characterization by transmission electron microscopy. (B) Molecularcharacterization by western blot analysis. Protein extracts prepared from serum (S) or exosomes (Ex) were assessed using antibodies againstexosomal protein markers (TSG101, CD63, CD9, HSP90α and Flotillin).

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(Figure 2B), indicating a clear distinction between theMHFMD, ESHFMD, and control (Figure 2C). Four ofthese miRNAs were up-regulated and seven were down-regulated in MHFMD and ESHFMD serum samplescompared to the control. miR-671-5p was only ap-peared in healthy children and MHFMD, and was al-most undetectable in ESHFMD patients. These data canprovide a valuable repertoire to discover miRNA-basedbiomarkers for distinguishing ESHFMD from MHFMD.

qRT-PCR verification of miRNA expressionThe most differentially expressed miRNAs miR-671-5p,miR-16-5p, miR-150-3p, and miR-4281 (results frommicroarray analysis) were selected and tested using an in-dependent cohort of 54 exosome samples (18 ESHFMD,18 MHFMD, and 18 healthy control) subjected to qRT-PCR; all the miRNAs passed the quality control. miR-642a-3p expression was proposed as the normalizationcontrol for exosomal miRNA levels, as the expression levelof miR-642a-3p was almost identical among control,MHFMD, and ESHFMD groups with almost no differ-ences in raw Ct values, which was consistent with themicroarray analysis results.

Result indicated that the miR-671-5p, miR-16-5p, andmiR-150-3p expression levels were significantly differentbetween MHFMD or ESHFMD and the control; more-over, miR-671-5p was almost undetectable in ESHFMDin contrast to MHFMD and the control. miR-16-5p,miR-150-3p, and miR-671-5p showed the same changesas in the microarray analysis. It obtained that the miR-16-5p expression in exosome in HFMD serum sampleswere found especially higher than that in the normalchildren. In contrast, miR-671-5p and miR-150-3p levelswere lower in HFMD than in controls. The serum miR-16-5p level increased by 5.98 and 10.31 fold in MHFMDand ESHFMD patients, respectively. Whereas, miR-4281showed no significant differences between the ESHFMDgroup and the control group, or between the ESHFMDgroup and MHFMD group (Figure 3).

Evaluation of miR-671-5p, miR-16-5p, and miR-150-3p aspotential diagnostic markersTo determine whether serum miRNA levels in exsomecan be used to distinguish patients with ESHFMD fromthose with MHFMD or controls, we established ROCcurves to analyse the difference in miR-671-5p, miR-16-

Figure 2 Microarray analysis of HFMD. (A) Scatter plot of miRNA expression determined by miRNA microarray analysis. The expression profileof 1,887 miRNAs on a log2 scale in HFMD and in the control group is plotted. Red and green dots represent the number of miRNAs that weresignificantly (P < 0.05 and 1.5 fold-change cut-off) up-regulated and down-regulated, respectively, in HFMD, and black dots represent a lack ofdifferential expression. (a) Comparison between normal controls and MHFMD samples, (b) comparison between normal controls and ESHFMDsamples, and (c) comparison between MHFMD and ESHFMD samples. (B) Venn diagram illustrates the overlapping results of the differentiallyexpressed miRNAs in MHFMD vs. control, ESHFMD vs. control, and MHFMD vs. ESHFMD comparisons. Circles include numbers of up-regulatedor down-regulated human miRNAs of each pair-wise comparison. The 11 miRNAs in the centre of the Venn diagram represent miRNAs that aredifferentially expressed in all group comparisons. (C) Heat map analysis of differentially regulated miRNAs among ESHFMD, MHFMD, and controlserum samples according to Venn diagram analysis. Heat map colours represent relative miRNA expression as indicated in the colour key. Redrepresents high expression, whereas green represents low expression.

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5p, and miR-150-3p serum levels between groups. Com-paring the MHFMD and control groups, the ROC curveareas for miR-671-5p, miR-16-5p, and miR-150-3p werefound to be 0.79 (95% CI, 0.62–0.91), 0.80 (95% CI,0.63–0.91), and 0.89 (95% CI, 0.74–0.97), respectively.The specificity and the sensitivity of each of these miR-NAs were 72% and 82%, 72% and 83%, and 100% and78%, for the MHFMD and control groups (Figure 4A),respectively. These results clearly show that miR-671-5p,miR-16-5p, and miR-150-3p serum levels can distinguishMHFMD from healthy controls.

We next compared the serum levels of these miRNAsbetween ESHFMD and control groups. The ROC curveareas of miR-671-5p, miR-16-5p, and miR-150-3p were1.00 (95% CI, 0.90–1.00), 0.98 (95% CI, 0.86–1.00), and0.83 (95% CI, 0.67–0.93), respectively. The specificity andthe sensitivity for these miRNAs were 100% and 100%,100% and 89%, and 100% and 78%, respectively, in theESHFMD and control groups (Figure 4B). These resultsalso demonstrate that the levels of these three miRNAs(miR-671-5p, miR-16-5p, and miR-150-3p) can distinguishESHFMD from healthy controls.

Figure 3 The box plots depict the relative expression level of four miRNAs (miR-671-5p, miR-150-3p, miR-16-5p and miR-4281)assessed by qRT-PCR in ESHFMD, MHFMD, and control serum samples. Statistically significant differences were determined using unpairedStudent’s t-test.

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The comparison of ESHFMD with MHFMD indicatedthat miR-671-5p, miR-16-5p, and miR-150-3p levels areuseful markers for discriminating patients with ESHFMDfrom those with MHFMD because the ROC curve area ofmiR-671-5p, miR-16-5p, and miR-150-3p was 0.82 (95%

Figure 4 Receiver operating characteristic (ROC) curve analysis of miRMHFMD from the control (A), ESHFMD from the control (B), and MHF

CI, 0.65–0.92), 0.76 (95% CI, 0.59–0.88), and 0.76 (95% CI,0.58–0.88) and the specificity and the sensitivity were 83%and 78%, 78% and 89%, and 78% and 88% respectively, inthe two groups (Figure 4C). Together, these resultsdemonstrate that the miR-671-5p, miR-16-5p, and

-671-5p, miR-150-3p, miR-16-5p was performed to discriminateMD from ESHFMD (C).

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miR-150-3p serum levels can be used to distinguishMHFMD, ESHFMD, and control samples and reflectedstrong separation among these samples.

GO Terms and KEGG pathway annotation ofmiRNA targetsGO biology process and KEGG pathway enrichmentswere performed by mapping the predicted target genes,and 20 biology processes for each miRNA and 30, 18,and 2 KEGG pathways for miR-16-5p, miR-150-3p, andmiR-671-5p were annotated.The main GO categories annotation showed that devel-

opmental process and regulation of cellular process forthe putative target genes of miR-16-5p and miR-150-3p,and neurogenesis, regulation of nervous system develop-ment, neuromuscular process controlling balance and ner-vous system development for the putative target genes ofmiR-150-3p and miR-671-5p were the most significantlyenriched GO terms (Figure 5).The KEGG pathway enrichment analysis indicated that

the putative targets for these miRNAs were mainly in-volved in pathways such as those related pathways in neu-rotrophin signalling pathway, insulin signalling pathway,TGF-beta signalling pathway, and MAPK signalling path-way (Figure 5).

Figure 5 GO category for putative target genes. P < 0.05 was used as aanalysis for putative target genes. P < 0.05 was used as a threshold to selecP-value. (A) miR-16-5p, (B) miR-150-3p, and (C) miR-671-5p.

DiscussionEarly and rapid separation of different disease infectioncondition may benefit controlling and prognosis prediction.Against HFMD, many substantial progresses in understand-ing the biology and pathogenesis agents continues [6,7,30].As ESHFMD usually caused majority death, we need torecognize MHFMD from ESHFMD on the way to reducemortality. Early clinical diagnosis, such as disease-associatedmiRNAs in exosome could serve as biomarkers, has theability for understanding different infection states forHFMD. It could be also helpful for revealing some intri-guing aspects regarding the potential function of these miR-NAs in HFMD.In this study, we investigated the expression levels of

miRNA profile in exosome from serum samples inMHFMD and ESHFMD and healthy children. By the two-step screening and confirmation approach, we identified3 miRNAs (miR-671-5p, miR-16-5p, and miR-150-3p)which were significantly different in patients in com-parison to controls. To evaluate the efficiency of thesemiRNAs for diagnosing, ROC curves were constructedfor each miRNA. Expression levels of three miRNAs(miR-671-5p, miR-16-5p, and miR-150-3p) showed goodability to efficiently distinguish MHFMD or ESHFMDfrom healthy control, with AUC that ranged from 0.79 to1.00. Combination of three selected exsome miRNAs also

threshold to select significant GO categories and for KEGG pathwayt significant KEGG pathways; lgP is the negative logarithm of the

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created significantly increased the diagnosis efficiencyto distinguish ESHFMD from MHFMD with AUC of0.76 to 0.82. Furthermore, the miRNAs (miR-671-5p)was almost undetectable in ESHFMD when distinguish-ing it from MHFMD.Moreover, the miR-16-5p expression in exosome was

found especially higher, and miR-671-5p and miR-150-3p levels in exosome were particularly lower than that inhealthy children. Recent publications revealed that themiR-16-5p is up-regulated in various human diseases in-cluding Alzheimer’s disease and prion disease. It hasbeen identified as an important intercellular messengermediating amyloid precursor protein (APP) protein for-mation [31]. Up-regulation of miR-16-5p during theearly disease stage and decreased expression with diseaseprogression was also found in prion disease [31]. Itwould therefore be interesting to determine that themiR-16-5p may have a neuroprotective role. Moreover,the miR-150-3p levels may be negatively correlated withplasma TNF-α level in patients [32] and those miR-671-5p levels may regulate gene expression to promotetumour growth [33]. From all the related study above, itmay suggest that miR-16-5p, miR-150-3p and miR-671-5p in exosomes may play an important role in the move-ment of HFMD.Further investigate the possible functions of miRNAs

through GO terms and KEGG pathway annotation. Theputative targets for these miRNAs were mainly involvedin such as MAPK signalling pathway related to potentialantiviral mechanisms [34], and the neurotrophin signal-ling pathway considered to influence the expression ofAPP [35]. The predicted target gene of miR-16-5p wasCLU that could clear of beta amyloid peptide, which wasone of the major brain lesions with Alzheimer’s disease[36-38]. Moreover, the polymorphic genes associatedwith Alzheimer’s disease delineate a clear pathway inyoung populations [39].

ConclusionsWhereas definitive diagnosis depends on organismspeci-fic detection results, our data recommended that exso-mal miRNA profile provide a supplemental biomarkerfor differential infection stage at an early stage. Theymay share to several signalling pathways, for instance,the MAPK signalling pathway and neurotrophin signal-ling pathway, influenced by such as APP formation pro-tein expression. Further studies, including the functionalexploration of exosomal miRNA profile, will be neededto make these hypotheses served as the clinical diagnosisbiomarkers.

Competing interestsThe authors declare that they have no competing interests.

Authors’ contributionsLD, HLJ and CWL created the concept and design of this study. CHH, GQYparticipated in sample diagnosis and collection. YFX and LJM performed theexperiments. HLJ and ZYW were responsible for the statistical analysis. LD,HLJ and CWL drafted, revised and edited the manuscript. All authors readand approved the final manuscript.

AcknowledgementsThe work was supported by National Clinical Key Specialty ProjectFoundation, Guangdong Natural Science Foundation (9151008901000033to L.D.) and Applied Basic Research Program of Technology and InformationBureau Guangzhou City (2013 J4100022 to CH.H.).

List of Support/Grant Information, including location(city/state/country)This work was supported by National Clinical Key Specialty ProjectFoundation, Guangdong Natural Science Foundation (9151008901000033)and Applied Basic Research Program of Technology and Information BureauGuangzhou City (2013 J4100022).

Author details1Guangzhou Women and Children’s Medical Center, Guangzhou 510120,Guangdong, China. 2Key Laboratory of Functional Protein Research ofGuangdong Higher Education Institutes, Institute of Life and HealthEngineering, College of Life Science and Technology, Jinan University,Guangzhou 510632, Guangdong, China. 3Guangdong Institute ofMicrobiology/State Key Laboratory of Applied Microbiology Southern China/Guangdong Provincial Key Laboratory of Microbial Culture Collection andApplication, Guangzhou 510070, Guangdong, China. 4Guangdong ProvincialKey Laboratory of Pharmaceutical Bioactive Substances, School of BasicCourses, Guangdong Pharmaceutical University, Guangzhou 510006,Guangdong, China.

Received: 9 August 2014 Accepted: 11 September 2014Published: 17 September 2014

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doi:10.1186/1471-2334-14-506Cite this article as: Jia et al.: MicroRNA expression profile in exosomediscriminates extremely severe infections from mild infections for hand,foot and mouth disease. BMC Infectious Diseases 2014 14:506.

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